Cotton variety phy333wrf

ABSTRACT

The disclosure relates to a cotton variety, designated PHY333WRF, the plants and seeds of the cotton variety PHY333WRF, methods for producing a cotton plant, either varietal or hybrid, produced by crossing the cotton variety PHY333WRF with itself or with another cotton plant, hybrid cotton seeds and plants produced by crossing the variety PHY333WRF with another cotton variety or plan, methods for producing a cotton plant containing in its genetic material one or more transgenes, and the transgenic cotton plants produced by that method. This disclosure also relates to cotton varieties derived from cotton variety PHY333WRF, to methods for producing other cotton varieties derived from cotton variety PHY333WRF, and to the varieties derived by the use of those methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/911,181, which was filed in the U.S. Patent and Trademark Office onDec. 3, 2013, the entirety of which is expressly incorporated byreference herein.

FIELD OF THE INVENTION

This invention is in the field of cotton breeding.

BACKGROUND OF THE INVENTION

Cotton (Gossypium spp.) is the world's most important textile fiber cropand is one of the world's most important oilseed crops. Cotton plantsprovide a source of human food, livestock feed, and raw material inindustry. Cotton seed is pressed for cooking oil and the residualcottonseed oil meal is used for animal feed. Industrial uses of cottoninclude candle wicks, twine, paper and a multitude of fabric products.

The genus Gossypium is very large, currently containing more than 50species. Two tetraploid species of Gossypium have spinnable seed fiberscalled lint. These two species are G. hirsutum (referred to as AmericanUpland cotton) and G. barbadense (referred to as Pima cotton).

The goal of a cotton breeder is to improve a cotton plant's performanceand therefore, its economic value by combining various desirable traitsinto a single plant. Improved performance is manifested in many ways.Higher yields of cotton plants contribute to increased lint fiberproduction, more profitable agriculture and lower cost of products forthe consumer. Improved plant health increases the yield and quality ofthe plant and reduces the need for application of protective chemicals.Adapting cotton plants to a wider range of production areas achievesimproved yield and vegetative growth. Improved plant uniformity enhancesthe farmer's ability to mechanically harvest cotton.

Cotton is a dicot plant with perfect flowers, i.e., cotton has male,pollen-producing organs and separate female, pollen receiving organs onthe same flower. The cultivated cotton flower is surrounded by threetriangular bracts forming what is commonly known as squares. The flowercontains an open corolla with five petals, a staminal column bearingclusters of stamens and forming a tube that encloses the style. Thecompound pistil consists of three to five carpels with stigmasprotruding above the anthers. The ovary develops into a three- tofive-loculed capsule or boll. From seven to nine seeds are set withineach lock or locule. On the day preceding anthesis, a twisted corollaemerges from the square. On the day of anthesis, the corolla opens andpollen shedding occurs. The corolla turns red the day following anthesisand later falls from the plant. Pollination occurs with the opening ofthe anthers and shedding of pollen on the stigma or with the deposit ofpollen on the stigma by insects.

Because cotton has both male and female organs on the same flower,cotton breeding techniques take advantage of the plant's ability to bebred by both self-pollination and cross-pollination. Self-pollinationoccurs when pollen from the male organ is transferred to a female organon the same flower on the same plant. Self-incompatibility is a form ofinfertility caused by the failure of cotton plants with normal pollenand ovules to set seed due to some physiological hindrance that preventsfertilization. Self-incompatibility restricts self-pollination andinbreeding and fosters cross-pollination. Cross-pollination occurs whenpollen from the male organ on the flower of one plant is transferred toa female organ on the flower on a different plant.

A plant is sib-pollinated (a type of cross-pollination) when individualswithin the same family or line are used for pollination (i.e. pollenfrom a family member plant is transferred to the stigmas of anotherfamily member plant). Self-pollination and sib-pollination techniquesare traditional forms of inbreeding used to develop new cottonvarieties, but other techniques exist to accomplish inbreeding. Newcotton varieties are developed by inbreeding heterozygous plants andpracticing selection for superior plants for several generations untilsubstantially homozygous plants are obtained. During the inbreedingprocess with cotton, the vigor of the lines decreases and after asufficient amount of inbreeding, additional inbreeding merely serves toincrease seed of the developed variety. Cotton varieties are typicallydeveloped for use in the production of hybrid cotton lines.

Natural, or open pollination, occurs in cotton when bees or otherinsects transfer pollen from the anthers to the stigmas and can includeboth self- and cross-pollination. Such pollination is accomplishedalmost entirely by the bees or other pollinating insects as the pollenis heavy and sticky and accordingly, interplant transfer of pollen bythe wind is of little importance. Vigor is restored when two differentvarieties are cross-pollinated to produce the first generation (F₁)progeny. A cross between two defined substantially homozygous cottonplant varieties always produces a uniform population of heterozygoushybrid cotton plants and such hybrid cotton plants are capable of beinggenerated indefinitely from the corresponding variety cotton seedsupply.

When two different, unrelated cotton parent plant varieties are crossedto produce an F₁ hybrid, one parent variety is designated as the male,or pollen parent, and the other parent variety is designated as thefemale, or seed parent. Because cotton plants are capable ofself-pollination, hybrid seed production requires elimination of orinactivation of pollen produced by the female parent to render thefemale parent plant male sterile. This serves to prevent the cottonplant variety designated as the female from self-pollinating. Differentoptions exist for controlling male fertility in cotton plants such asphysical emasculation, genetic male sterility, cytoplasmic malesterility and application of gametocides. Incomplete removal of maleparent plants from a hybrid seed production field before harvestprovides the potential for unwanted production of self-pollinated orsib-pollinated seed, which can be unintentionally harvested and packagedwith hybrid seed.

The development of new cotton plant varieties and hybrid cotton plantsis a slow, costly interrelated process that requires the expertise ofbreeders and many other specialists. The development of new varietiesand hybrid cotton plants in a cotton plant breeding program involvesnumerous steps, including: (1) selection of parent cotton plants(germplasm) for initial breeding crosses; (2) inbreeding of the selectedplants from the breeding crosses for several generations to produce aseries of varieties, which individually breed true and are highlyuniform; and (3) crossing a selected variety with an unrelated varietyto produce the F₁ hybrid progeny having restored vigor.

Cotton plant varieties and other sources of cotton germplasm are thefoundation material for all cotton breeding programs. Despite theexistence and availability of numerous cotton varieties and other sourcegermplasm, a continuing need still exists for the development ofimproved germplasm because existing parent cotton varieties lose theircommercial competitiveness over time. Embodiments of the presentdisclosure addresses this need by providing a novel cotton inbredvariety designated PHY333WRF that possesses broad adaptation andexcellent yield stability in the full-maturity cotton growing regions ofthe US; excellent fiber properties such as micronaire, length, strength(g/tex), and fiber uniformity; the WideStrike® transgenic events (Cry1Fand Cry1Ac from Bacillus thuringiensis) for resistance to Lepidopterainsects; and the Roundup Ready® Flex transgenic event for tolerance toglyphosate herbicide. PHY333WRF contributes such characteristics tohybrids relative to other similar hybrids in the same maturity groups.To protect and to enhance yield production, trait technologies and seedtreatment options provide additional crop plan flexibility and costeffective control against insects, weeds and diseases, thereby furtherenhancing the potential of this variety and hybrids with PHY333WRF as aparent.

SUMMARY OF THE INVENTION

Embodiments of this disclosure relate to a cotton variety designatedPHY333WRF that includes plants and seeds of cotton variety PHY333WRF.Further embodiments relate to lint having novel characteristics whetheror not produced by the claimed cotton variety. Methods for producingcotton plants, such as cotton plant varieties, hybrid cotton plants, orother cotton plants, as by crossing cotton variety PHY333WRF with itselfor any different cotton plant are an integral part of certainembodiments, as are the resultant cotton plants including the plantparts and seeds. Other embodiments relate to methods for producingPHY333WRF-derived cotton plants, to methods for producing male sterilePHY333WRF cotton plants, e.g., cytoplasmic male sterile PHY333WRF cottonplants and to methods for regenerating such plants from tissue culturesof regenerable cells as well as the plants obtained therefrom. Methodsfor producing a cotton plant containing in its genetic material one ormore transgenes, and the transgenic cotton plants produced by thatmethod, are also a part of further embodiments.

In one embodiment, the present disclosure relates to a seed of thecotton variety designated PHY333WRF, or a part thereof, representativeseed of the variety having been deposited under ATCC Accession No.PTA-120742 on Dec. 2, 2013. In a further aspect, the disclosure relatesto a part of this seed, selected from the group consisting of hull (seedcoat), germ and endosperm. In a further aspect, the disclosure relatesto this seed, further comprising a coating. In a further aspect, thedisclosure relates to a substantially homogenous composition of thisseed.

In another embodiment, the present disclosure relates to a method forproducing a seed of a cotton plant, comprising: (a) planting seed of thecotton variety designated PHY333WRF in proximity to itself or todifferent seed from a same variety; (b) growing plants from the seedunder pollinating conditions; and (c) harvesting the resultant seed. Ina further aspect, the disclosure relates to a cotton seed produced bythis method. In a further aspect, the disclosure relates to this method,further comprising pre-treating the seed before performing step (a). Ina further aspect, the disclosure relates to this method, furthercomprising treating the growing plants or soil surrounding the growingplants with an agricultural chemical.

In another embodiment, the present disclosure relates to a cotton plantproduced by growing a seed of the cotton variety designated PHY333WRF.In a further aspect, the disclosure relates to a part of this cottonplant, selected from the group consisting of an intact plant cell, aplant protoplast, embryos, pollen, flowers, seeds, linters, fibers,pods, gossypol glands, leaves, bolls, stems, roots, root tips, andanthers. In a further aspect, the disclosure relates to fibers of thisplant. In a further aspect, the disclosure relates to staples of thisplant. In a further aspect, the disclosure relates to a cotton plant, ora part thereof, having all the physiological and morphologicalcharacteristics of this cotton plant. In a further aspect, thedisclosure relates to a substantially homogenous population of thesecotton plants. In a further aspect, the disclosure relates to thissubstantially homogenous population of cotton plants, wherein thepopulation is present in a field and the field further comprises other,different cotton plants.

In another embodiment, the present disclosure relates to a method forproducing a cotton plant, comprising: (a) crossing cotton variety plantPHY333WRF, representative seed of the cultivar having been depositedunder ATCC Accession No. PTA-120742 on Dec. 2, 2013, with anotherdifferent cotton plant to yield progeny cotton seed. In a furtheraspect, the disclosure relates to this method, wherein the other,different cotton plant is a cotton variety. In a further aspect, thedisclosure relates to this method, further comprising: (b) growing theprogeny cotton seed from step (a) under self-pollinating orsib-pollinating conditions for about 5 to about 7 generations; and (c)harvesting resultant seed. In a further aspect, the disclosure relatesto this method, further comprising selecting plants obtained fromgrowing at least one generation of the progeny cotton seed for adesirable trait.

In another embodiment, the present disclosure relates to a method ofintroducing a desired trait into cotton variety PHY333WRF,representative seed of the variety having been deposited under ATCCAccession No. PTA-120742 on Dec. 2, 2013, comprising: (a) crossingPHY333WRF plants with plants of another cotton variety that comprise adesired trait to produce F1 progeny plants; (b) selecting F1 progenyplants that have the desired trait; (c) crossing selected progeny plantswith PHY333WRF plants to produce backcross progeny plants; (d) selectingfor backcross progeny plants that comprise the desired trait andphysiological and morphological characteristics of cotton varietyPHY333WRF; and (e) performing steps (c) and (d) one or more times insuccession to produce the selected or higher backcross progeny plantsthat comprise the desired trait and all of the physiological andmorphological characteristics of cotton variety PHY333WRF listed inTable 1 as determined at the 5% significance level when grown in thesame environmental conditions. In a further aspect, the disclosurerelates to this method, wherein the plants of the other cotton varietycomprise a desired trait selected from the group consisting of malesterility, drought tolerance, herbicide resistance, insect resistance,and resistance to bacterial, fungal and viral disease. In a furtheraspect, the disclosure relates to this method, further comprising usingdirect or indirect selection to determine whether the desired trait ispresent in a progeny plant.

In another embodiment, the present disclosure relates to a method forproducing a cotton plant, comprising: (a) crossing a cotton plantproduced by growing a seed of the cotton variety designated PHY333WRFwith another different cotton plant to produce a diploid or progenyplant; (b) generating a haploid progeny plant from the diploid progenyplant; (c) generating a diploid plant from the haploid progeny plant;and (d) selecting the diploid cotton plant. In a further aspect, thedisclosure relates to this method, wherein the haploid progeny plant isgenerated by culturing a haploid explant from the diploid progeny plant.In a further aspect, the disclosure relates to this method, wherein thehaploid progeny plant is generated by crossing the progeny plant withanother, different plant that induces haploid cotton plants. In afurther aspect, the disclosure relates to this method, wherein theother, different plant is a cotton plant that comprises ahaploid-inducing gene. In a further aspect, the disclosure relates tothis method, wherein the diploid plant of step (c) is generated bysubjecting the haploid progeny plant to a treatment that induceschromosome doubling in the cultured explant. In a further aspect, thedisclosure relates to this method, wherein the diploid plant of step (c)is generated by self-pollinating the haploid progeny plant.

In another embodiment, the present disclosure relates to a method forproducing a cotton plant, comprising: (a) inducing a mutation in acotton plant produced by growing a seed of the cotton variety designatedPHY333WRF, or a part thereof; and, (b) selecting mutated cotton plants.In a further aspect, the disclosure relates to this method, wherein themutation is artificially induced by a method selected from the groupconsisting of elevated temperature, long-term seed storage, tissueculture conditions, radiation, and chemical mutagenesis.

In another embodiment, the present disclosure relates to a method forproducing a cotton plant variety, comprising: (a) growing firstgeneration hybrid cotton plants having PHY333WRF, representative seed ofthe variety having been deposited under ATCC Accession No. PTA-120742 onDec. 2, 2013, as a parent cotton plant; (b) inbreeding the firstgeneration hybrid cotton plants or crossing the first generation hybridcotton plants with different cotton plants to yield progeny cotton seed;(c) growing the progeny cotton seed of step (b) to yield further progenycotton seed; (d) repeating the inbreeding or the crossing and thegrowing steps of (b) and (c) from about 0 to about 7 times to generatecotton varietal plants. In a further aspect, the disclosure relates to acotton plant variety produced by this method.

In another embodiment, the present disclosure relates to a method forproducing cotton variety PHY333WRF, representative seed of the varietyhaving been deposited under ATCC Accession No. PTA-120742 on Dec. 2,2013, comprising: (a) planting a collection of seed comprising seed of ahybrid, one of whose parents is PHY333WRF, the collection alsocomprising seed of the variety PHY333WRF; (b) growing plants from thecollection of seed; (c) identifying a varietal parent plant; (d)controlling pollination in a manner that preserves the homozygosity ofthe varietal parent plant; and, (e) harvesting the resultant seed fromthe identified varietal parent plant which was pollinated to preserveits homozygosity. In a further aspect, the disclosure relates to thismethod, wherein step (c) comprises identifying plants with decreasedvigor. In a further aspect, the disclosure relates to a method forproducing a varietal cotton plant comprising: sib-pollinating plantsobtained by growing the harvested resultant seed of step (e) of thismethod. In a further aspect, the disclosure relates to a method forproducing a varietal cotton plant comprising: crossing PHY333WRF cottonplants with cotton plants obtained by growing the hybrid seed of step(a) of this method.

In another embodiment, the present disclosure relates to a method forproducing a hybrid cotton seed comprising crossing a first varietalparent cotton plant with a second varietal parent cotton plant andharvesting resultant hybrid cotton seed, wherein the first varietalcotton plant or the second varietal cotton plant is a cotton plantproduced by growing a seed of the cotton variety designated PHY333WRF.

In another embodiment, the present disclosure relates to a method forproducing a hybrid cotton seed comprising the steps of: (a) planting inpollinating proximity seeds of a first and a second varietal parentcotton plants, wherein the first varietal cotton plant or the secondvarietal cotton plant is a cotton plant produced by growing a seed ofthe cotton variety designated PHY333WRF; (b) cultivating the seeds ofthe first and the second varietal cotton plants into plants that bearflowers; (c) controlling the male fertility of the first or the secondvarietal cotton plant to produce a male sterile cotton plant; (d)allowing cross-pollination to occur between the first and secondvarietal cotton plants; and, (e) harvesting seeds produced on the malesterile cotton plant. In a further aspect, the disclosure relates tothis method, wherein the varietal cotton plant that is the cotton plantproduced by growing a seed of the cotton variety designated PHY333WRF isa female parent. In a further aspect, the disclosure relates to thismethod, wherein the varietal cotton plant that is the cotton plantproduced by growing a seed of the cotton variety designated PHY333WRF isa male parent. In a further aspect, the disclosure relates to a hybridcotton seed produced by this method. In a further aspect, the disclosurerelates to a hybrid cotton plant, or parts thereof, producing by growingthis hybrid cotton seed. In a further aspect, the disclosure relates toa tissue culture of regenerable cells from this hybrid cotton plant. Ina further aspect, the disclosure relates to a cotton seed obtained bygrowing the hybrid cotton seed produced by this method and harvestingthe resultant cotton seed from produced plants.

In another embodiment, the present disclosure relates to a method forproducing a hybrid cotton seed comprising crossing a first varietalparent cotton plant with a second varietal parent cotton plant andharvesting the resultant hybrid cotton seed, wherein the first varietalcotton plant or the second varietal cotton plant is a progeny plant of across of the cotton plant produced by growing a seed of the cottonvariety designated PHY333WRF and another varietal cotton plant. In afurther aspect, the disclosure relates to a hybrid cotton seed producedby this method. In a further aspect, the disclosure relates to a hybridcotton plant, or a part thereof, produced by growing this hybrid cottonseed. In a further aspect, the disclosure relates to a cotton seedproduced by growing this hybrid cotton plant and harvesting theresultant cotton seed.

In another embodiment, the present disclosure relates to an F1 hybridseed produced by crossing the varietal cotton plant produced by growinga seed of the cotton variety designated PHY333WRF with another,different cotton plant. In a further aspect, the disclosure relates to ahybrid cotton plant, or a part thereof, produced by growing this hybridcotton seed. In a further aspect, the disclosure relates to this hybridcotton seed, wherein the other, different plant is not a member of thehirsutum species. In a further aspect, the disclosure relates to thishybrid cotton seed, wherein the other, different plant is a member ofthe barbadense species. In a further aspect, the disclosure relates tothis hybrid cotton seed, wherein the other, different plant is a memberof a genus Gossypium. In a further aspect, the disclosure relates tothis hybrid cotton seed, wherein the other, different plant is a memberof the family Malvaceae.

In another embodiment, the present disclosure relates to a method forproducing a PHY333WRF-derived cotton plant, comprising: (a) crossingcotton variety PHY333WRF, representative seed of the variety having beendeposited under ATCC Accession No. PTA-120742 on Dec. 2, 2013, with asecond cotton plant to yield progeny cotton seed; and (b) growing saidprogeny cotton seed, under plant growth conditions, to yield thePHY333WRF-derived cotton plant. In a further aspect, the disclosurerelates to a PHY333WRF-derived cotton plant, or a part thereof, producedby this method. In a further aspect, the disclosure relates to thismethod, further comprising: (c) crossing the PHY333WRF-derived cottonplant with itself or another cotton plant to yield additionalPHY333WRF-derived progeny cotton seed; (d) growing the progeny cottonseed of step (c) under plant growth conditions, to yield additionalPHY333WRF-derived cotton plants; and (e) repeating the crossing andgrowing steps of (c) and (d) from 0 to 7 times to generate furtherPHY333WRF-derived cotton plants. In a further aspect, the disclosurerelates to this method, still further comprising utilizing plant tissueculture methods and/or haploid breeding to derive progeny of thePHY333WRF-derived cotton plant.

In another embodiment, the present disclosure relates to a tissueculture of regenerable cells from the cotton plant produced by growing aseed of the cotton variety designated PHY333WRF. In a further aspect,the disclosure relates to this tissue culture, the cells or protoplastsof the tissue culture being from a tissue selected from the groupconsisting of embryos, pollen, flowers, seeds, linters, fibers, pods,gossypol glands, leaves, bolls, stems, roots, root tips, and anthers. Ina further aspect, the disclosure relates to a cotton plant regeneratedfrom this tissue culture, wherein the regenerated plant expresses allthe morphological and physiological characteristics of varietyPHY333WRF.

In another embodiment, the present disclosure relates to a cotton plantwith all of the physiological and morphological characteristics ofcotton variety PHY333WRF, wherein the cotton plant is produced by atissue culture process using the cotton plant produced by growing a seedof the cotton variety designated PHY333WRF as a starting material forthe process.

In another embodiment, the present disclosure relates to a method forregenerating a cotton plant comprising the steps of: (a) culturing anexplant comprising a tissue selected from the group consisting of atissue obtained from cotton plant variety PHY333WRF, representative seedhaving been deposited under ATCC Accession No. PTA-120742 on Dec. 2,2013, an immature tissue obtained from a hybrid cotton plant havingPHY333WRF as a parent, and a PHY333WRF-derived cotton plant; and, (b)initiating regeneration. In a further aspect, the disclosure relates tothis method, wherein the explant is an immature tissue.

In another embodiment, the present disclosure relates to a cotton plantproduced by growing a seed of the cotton variety designated PHY333WRF,wherein the PHY333WRF plant is rendered male sterile. In a furtheraspect, the disclosure relates to this cotton plant, wherein the malesterile PHY333WRF plant is a cytoplasmic male sterile plant.

In another embodiment, the present disclosure relates to a method forproducing a male sterile PHY333WRF cotton plant, comprising: (a)crossing a varietal cotton plant produced by growing a seed of thecotton variety designated PHY333WRF, with a cytoplasmic male sterilecotton plant that generates haploids; (b) identifying haploid plants;and, (c) crossing the haploid plants with the varietal cotton plantPHY333WRF to produce male sterile PHY333WRF cotton plants.

In another embodiment, the present disclosure relates to a cotton plant,or a part thereof, produced by growing a seed of the cotton varietydesignated PHY333WRF, wherein the plant or part thereof has beentransformed so that its genetic material contains one or more transgenesoperably linked to one or more regulatory elements. In a further aspect,the disclosure relates to a method for producing a cotton plant thatcontains in its genetic material one or more transgenes, comprisingcrossing this cotton plant with either a second plant of another cottonvariety, or a non-transformed cotton plant of the variety PHY333WRF, sothat the genetic material of the progeny that result from the crosscontains the transgene(s) operably linked to a regulatory element. In afurther aspect, the disclosure relates to a cotton plant, or a partthereof, produced by this method.

In another embodiment, the present disclosure relates to a cotton plantproduced by growing a seed of the cotton variety designated PHY333WRF,or a part thereof, further comprising one or more transgenes. In afurther aspect, the disclosure relates to a seed of this plant. In afurther aspect, the disclosure relates to this cotton plant, wherein theone or more transgenes comprise a gene conferring upon said cotton plantinsect resistance, disease resistance or virus resistance. In a furtheraspect, the disclosure relates to this cotton plant, wherein the geneconferring upon the cotton plant insect resistance is a Bacillusthuringiensis gene.

In another embodiment, the present disclosure relates to a cotton plantproduced by growing a seed of the cotton variety designated PHY333WRF,or a part thereof, wherein the plant or a parts thereof has beentransformed so that its genetic material contains one or more transgenesoperably linked to one or more regulatory elements. In a further aspect,the disclosure relates to this cotton plant, wherein the one or moretransgenes comprise a gene conferring upon the cotton plant tolerance toan herbicide. In a further aspect, the disclosure relates to this cottonplant, wherein the herbicide is glyphosate, glufosinate, a phenoxy, asulfonylurea or an imidazolinone herbicide, a hydroxyphenylpyruvatedioxygenase inhibitor or a protoporphyrinogen oxidase inhibitor.

In another embodiment, the present disclosure relates to a method forproducing a population of PHY333WRF progeny cotton plants comprising:(a) obtaining a first generation progeny cotton seed from a plantproduced by growing a seed of the cotton variety designated PHY333WRF asa parent; (b) growing the first generation progeny cotton seed toproduce F₁ generation cotton plants and obtaining self or sib pollinatedseed from the F₁ generation cotton plants; and (c) producing successivefilial generations to obtain a population of PHY333WRF progeny cottonplants. In a further aspect, the disclosure relates to the population ofPHY333WRF progeny cotton plants produced by this method, the population,on average, deriving 50% of its alleles from PHY333WRF.

In another embodiment, the present disclosure relates to lint havingsubstantially the same characteristics of the lint produced by cottonvariety designated PHY333WRF, representative seed of the variety havingbeen deposited under ATCC Accession No. PTA-120742 on Dec. 2, 2013.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions of PlantCharacteristics

In the description and examples that follow, a number of terms are used.To provide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

Alternaria macrospora: This represents a visual assessment of the cottonplants for resistance to Alternaria leaf spot (Alternaria macrospora)rated as 0=not tested, 1=susceptible, or 2=moderately susceptible,3=moderately resistant, or 4=resistant. causes Alternaria leaf spot

Anthracnose: This represents a visual assessment of the cotton plantsfor resistance to Anthrancnose (Colletotrichum spp.) rated as 0=nottested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Area(s) of Adaptation: This represents whether the cotton plant isadapted (A), not adapted (NA) or not tested (NT) for the followingareas: Eastern, Delta, Central, Blacklands, Plains, Western, Arizona,and San Joaquin Valley.

Ascochyta Blight: This represents a visual assessment of the cottonplants for resistance to Ascochyta blight (Ascochyta gossypii) rated as0=not tested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Bacterial Blight (Race 1): This represents a visual assessment of thecotton plants for resistance to bacterial blight (race 1) (Xanthomonasmalvacearum) rated as 0=not tested, 1=susceptible, or 2=moderatelysusceptible, 3=moderately resistant, or 4=resistant.

Bacterial Blight (Race 2): This represents a visual assessment of thecotton plants for resistance to bacterial blight (race 2) (Xanthomonasmalvacearum) rated as 0=not tested, 1=susceptible, or 2=moderatelysusceptible, 3=moderately resistant, or 4=resistant.

Boll Breadth: This represents a comparison of the boll width at itsmiddle and its base rated as broadest at base, or broadest at middle.

Boll Shape: This represents the shape of the boll rated as length lessthan width, length equal to width, or length more than width.

Boll Type: This represents the boll type rated as stormproof, stormresistant, or open.

Boll Weevil: This represents a visual assessment of the cotton plantsfor resistance to Boll Weevil (Anthonomous grandis) rated as 0=nottested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Bollworm: This represents a visual assessment of the cotton plants forresistance to Bollworm (Helicoverpa zea) rated as 0=not tested,1=susceptible, or 2=moderately susceptible, 3=moderately resistant, or4=resistant.

Calyx Lobe: This represents the gossypol gland density on the calyx loberated as absent (normal), sparse, or more than normal.

Cotton Aphid: This represents a visual assessment of the cotton plantsfor resistance to Cotton Aphid (Aphis gossypii) rated as 0=not tested,1=susceptible, or 2=moderately susceptible, 3=moderately resistant, or4=resistant.

Cotton Fleahopper: This represents a visual assessment of the cottonplants for resistance to Cotton Fleahopper (Pseudatomoscellis seriatus)rated as 0=not tested, 1=susceptible, or 2=moderately susceptible,3=moderately resistant, or 4=resistant.

Cotton Leafworm: This represents a visual assessment of the cottonplants for resistance to Cotton Leafworm (Alabama argillacea) rated as0=not tested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Cutworm: This represents a visual assessment of the cotton plants forresistance to Cutworm (Agrotis ipsilon) rated as 0=not tested,1=susceptible, or 2=moderately susceptible, 3=moderately resistant, or4=resistant.

Days to 75% Open Bolls: This represents the number of days from plantinguntil which 75% of the bolls of a plant are open.

Diplodia Boll Rot: This represents a visual assessment of the cottonplants for resistance to Diplodia boll rot (Diplodia gossypina) rated as0=not tested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Distance to 1st Fruiting Branch: This represents the distance from thecotyledonary node to the first fruiting branch in centimeters.

Fall Armyworm: This represents a visual assessment of the cotton plantsfor resistance to Fall Armyworm (Spodoptera frugiperda) rated as 0=nottested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Fiber Elongation: This represents the amount that a fiber sample willstretch before breakage and is a measure of the deformation of thecotton fiber at rupture expressed as percent change in length based onthe original fiber length as measured by HVI.

Fiber Fineness: This represents a relative measure of size, diameter,linear density or weight per unit length expressed in terms of millitexor milligrams per tex unit.

Fiber Length: This represents fiber length expressed in hundredths of aninch as measured by High Volume Instrumentation (HVI).

Fiber Micronaire: This represents a measure of the fineness of thefiber. Within a cotton cultivar, micronaire is also a measure ofmaturity. Micronaire differences are governed by changes in perimeter orin cell wall thickness, or by changes in both. Within a variety, cottonperimeter is fairly constant and maturity will cause a change inmicronaire. Consequently, micronaire has a high correlation withmaturity within a variety of cotton. Maturity is the degree ofdevelopment of cell wall thickness. Micronaire often does not have agood correlation with maturity between varieties of cotton havingdifferent fiber perimeter. Micronaire values range from about 2.0 to 6.0and have the following meanings: below 2.9 very fine possible smallperimeter but mature (good fiber), or large perimeter but immature (badfiber); from 2.9 to 3.7 fine various degrees of maturity and/orperimeter; 3.8 to 4.6 average degree of maturity and/or perimeter; 4.7to 5.5 coarse usually fully developed (mature), but larger perimeter;and 5.6 or greater very coarse fully developed, large-perimeter fiber.

Fiber Strength: This represents the force required to rupture or tobreak a bundle of fibers as measured in grams per tex on the HVI.

Fiber Uniformity: This represents the uniformity of fiber length in asample as measured on the HVI, expressed as a percentage.

Fiber Yarn Tenacity: This represents the strength of a single strand ofyarn; the force required to break a yarn.

Foliage: This represents the general appearance of the plant leavesrated as sparse, intermediate, or dense.

Fruiting Branch: This represents fruiting pattern rated as clustered,short, or normal.

Fusarium Wilt: This represents a visual assessment of the cotton plantsfor resistance to Fusarium Wilt (Fusarium oxysporum) rated as 0=nottested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Gin Turnout: This represents the fraction of lint in a machine harvestedsample of seed cotton (lint, seed, and trash).

Glyphosate Herbicide Resistance: Resistance of a plant to the action ofglyphosate; conferred in crops by genetic transformation of the cropplant using a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genethat is insensitive to the effect of glyphosate, or a bacterialglyphosate oxidoreductase (GOX) gene that cleaves the nitrogen-carbonbond in glyphosate yielding aminomethylphosphonic acid.

Growth: This represents the growing pattern of the cotton plantfollowing a fruiting cycle rated as determinate, i.e., a completeinterruption of growth following a fruiting cycle, or indeterminate,i.e., a growth pattern in which stems continue to grow indefinitely.

Herbicide Resistance: When a plant has negligible effect from contactwith an herbicide because the plant does not take up the herbicide orsequesters the herbicide in a manner that renders it essentiallyharmless.

Insect Resistance: When a plant has negligible effect from contact witha potentially harmful insect because the plant has a biochemicalcomposition that repels, kills, or otherwise renders the insectessentially harmless to the plant.

Leaf Color: This represents a visual assessment of the leaf color of thecotton plant rated as greenish yellow, light green, medium green, darkgreen.

Leaf Glands: This represents the density of gossypol glands rated asabsent, sparse, normal, or more than normal.

Leaf Nectaries: This represents whether leaf nectaries are present orabsent on the uppermost fully expanded leaf.

Leaf Pubescence: This represents the density of leaf trichomes (“hairs”)on the bottom surface excluding veins of the uppermost fully expandedleaf rated as absent, sparse, medium, or dense in terms oftrichomes/cm².

Leaf Type: This represents the shape of the uppermost fully expandedleaf rated as normal, sub okra, okra, or super okra.

Lint Index: This represents the weight of lint per 100 seeds in grams.

Lint Percentage: This represents the lint (fiber) fraction of seedcotton (lint and seed).

Lint Yield: This represents the lint yield in pounds per acre.

Lygus: This represents a visual assessment of the cotton plants forresistance to Lygus (Lygus hesperus) rated as 0=not tested,1=susceptible, or 2=moderately susceptible, 3=moderately resistant, or4=resistant.

Mature Plant Height: This represents the height in centimeters of thecotton plant from the cotyledonary node to terminal.

Maturity (% Open Bolls): This represents the number of open bolls of aplant expressed as a percentage, generally measured about 2 weeks before100% of the bolls of a plant are open

Nodes to 1st Fruiting Branch: This represents the number of nodes fromthe cotyledonary node to the first fruiting branch, excluding thecotyledonary node.

Open Bolls: This represents the percentage of the bolls of a plant thatare open at harvest.

Petal Color: This represents a visual assessment of the petal colorrated as cream or yellow.

Petal Spot: This represents whether petal spot is present or absent onthe flowers of the cotton plant.

Phymatrotrichum Root Rot: This represents a visual assessment of thecotton plants for resistance to Phymatrotrichum root rot(Phymatrotrichum omnivore) rated as 0=not tested, 1=susceptible, or2=moderately susceptible, 3=moderately resistant, or 4=resistant.

Pink Bollworm: This represents a visual assessment of the cotton plantsfor resistance to Pink Bollworm (Pectinophora gossypiella) rated as0=not tested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Plant Habit: This represents the general growth habit of the plant ratedas spreading, intermediate or compact.

Pollen Color: This represents a visual assessment of pollen color ratedas cream or yellow.

Pythium: This represents a visual assessment of the cotton plants forresistance to Pythium (Pythium spp.) rated as 0=not tested,1=susceptible, or 2=moderately susceptible, 3=moderately resistant, or4=resistant.

Reniform Nematode: This represents a visual assessment of the cottonplants for resistance to Reniform Nematode (Rotylenchulus reniformis)rated as 0=not tested, 1=susceptible, or 2=moderately susceptible,3=moderately resistant, or 4=resistant.

Rhizoctonia solani: This represents a visual assessment of the cottonplants for resistance to boll rot (Rhizoctonia solani) rated as 0=nottested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Root-Knot Nematode: This represents a visual assessment of the cottonplants for resistance to Root-Knot Nematode (Meloidogyne incognita)rated as 0=not tested, 1=susceptible, or 2=moderately susceptible,3=moderately resistant, or 4=resistant.

Seed-Cotton Weight Per Boll: This represents the average number of gramsof seed cotton per boll on the cotton plant.

Seed Index: This represents the weight of 100 seeds in grams on a fuzzybasis.

Seeds Per Boll (Number): This represents the average number of seeds perboll on the cotton plant.

Southwestern Cotton Rust: This represents a visual assessment of thecotton plants for resistance to Southwestern Cotton Rust (Pucciniacacabata) rated as 0=not tested, 1=susceptible, or 2=moderatelysusceptible, 3=moderately resistant, or 4=resistant.

Spider Mite: This represents a visual assessment of the cotton plantsfor resistance to Spider Mite (Tetranychus spp.) rated as 0=not tested,1=susceptible, or 2=moderately susceptible, 3=moderately resistant, or4=resistant.

Stem Glands: This represents the density of gossypol glands rated asabsent, sparse, normal, or more than normal.

Stem Lodging: This represents the general appearance of the plant stemsrelative to their normal near vertical orientation rated as lodging,intermediate, or erect.

Stem Pubescence: This represents whether the stem pubescence isglabrous, intermediate, or hairy.

Stink Bug: This represents a visual assessment of the cotton plants forresistance to Stink Bug (Pitedia spp.; Euschistus spp.; Thyanta spp.)rated as 0=not tested, 1=susceptible, or 2=moderately susceptible,3=moderately resistant, or 4=resistant.

Thielaviopsis basicola: This represents a visual assessment of thecotton plants for resistance to black root rot (Thielaviopsis basicola)rated as 0=not tested, 1=susceptible, or 2=moderately susceptible,3=moderately resistant, or 4=resistant.

Thrips: This represents a visual assessment of the cotton plants forresistance to Thrips (Thrips spp.) rated as 0=not tested, 1=susceptible,or 2=moderately susceptible, 3=moderately resistant, or 4=resistant.

Tobacco Bud Worm: This represents a visual assessment of the cottonplants for resistance to Tobacco Budworm (Heliothis virescens) rated as0=not tested, 1=susceptible, or 2=moderately susceptible, 3=moderatelyresistant, or 4=resistant.

Verticillium Wilt: This represents a visual assessment of the cottonplants for resistance to (Verticillium dahliae) rated as 0=not tested,1=susceptible, or 2=moderately susceptible, 3=moderately resistant, or4=resistant.

II. Cotton Variety PHY333WRF

A. Cotton Plant PHY333WRF

In accordance with one aspect of the present disclosure, provided is anew Upland (Gossypium hirsutum) cotton seed and plants thereofdesignated PHY333WRF. Further embodiments relate to a method forproducing cotton seeds that includes, but is not limited to, the stepsof planting seed of cotton variety PHY333WRF in proximity to itself orto different seed from a same family or line, growing the resultingcotton plants under self-pollinating or sib-pollinating conditions withadequate isolation, and harvesting resultant seed obtained from suchplants using techniques standard in the agricultural arts that areuseful to bulk-up seed such as for hybrid production. Embodiments of thepresent disclosure also relate to varietal seed produced by such amethod.

In any cross between cotton plant variety PHY333WRF and another cottonplant variety, PHY333WRF can be designated as the male (pollen parent)or the female (seed parent). Optionally, the seed of cotton varietyPHY333WRF can be pre-treated to increase resistance of the seed and/orseedlings to stressed conditions, and further, the cotton plants orsurrounding soil can be treated with one or more agricultural chemicalsbefore harvest. Such agricultural chemicals can include herbicides,insecticides, pesticides and the like. Embodiments of the presentdisclosure also relate to a cotton plant that expresses substantiallyall of the physiological and morphological characteristics of cottonplant variety PHY333WRF and to a substantially homogenous population ofcotton plants having all the physiological and morphologicalcharacteristics of cotton plant variety PHY333WRF. Any cotton plantsproduced from cotton plant variety PHY333WRF are contemplated inembodiments of the present disclosure and are, therefore, within thescope thereof. A description of physiological and morphologicalcharacteristics of cotton plant PHY333WRF is presented in Table 1.

TABLE 1 Physiological and Morphological Characteristics of CultivarPHY333WRF Characteristic Value^(a) Area(s) of Adaptation Delta, EasternPlant Habit Intermediate Foliage Intermediate Stem Lodging IntermediateFruiting Branch Normal Growth Indeterminate Leaf Color Medium Green BollShape Length more than Width Boll Breadth Broadest at Middle Days afterplanting to 50% Open Bolls 121 Distance to 1st Fruiting Branch (cm) 27.8Nodes to 1st Fruiting Branch (number) 6.0 Mature Plant Height (cm) 90.8Leaf Type Normal Leaf Pubescence Intermediate Leaf Nectaries PresentStem Pubescence Intermediate Leaf Glands Normal Stem Glands Normal CalyxLobe Absent (Normal) Petal Color Cream Pollen Color Cream Petal SpotAbsent Seed Index (weight of 100 seeds in grams) 10.0 Lint Index (weightof 100 seeds in grams) 8.1 Lint Percentage 45.1 Seeds Per Boll (number)29.5 Seed-Cotton Weight Per Boll (grams) 4.9 Boll Type Open Fiber Length(hundredths of an inch) 116 Fiber Uniformity (percentage) 84.30 FiberStrength (grams per tex) 30.6 Fiber Elongation (percentage change) 6.8Fiber Micronaire 4.7 DISEASE AND INSECTS (1 = Susceptible, 2 =Moderately Susceptible, 3 = Moderately Resistant, 4 = Resistant)Fusarium Wilt 3 Root-Knot Nematode 1 TRANSGENE Insect Resistance Bt Cry1F and Bt Cry 1Ac Herbicide Resistance Glyphosate (MON 88913 event)^(a)These are typical values, which may vary due to the environment.Other values that are substantially equivalent are within the scope ofthis invention.

It will be appreciated by one having ordinary skill in the art that thevalues presented for the quantitative characteristics identified inTable 1 are typical values. These values can vary due to the environmentand accordingly, other values that are substantially equivalent are alsowithin the scope of embodiments of the disclosure.

Cotton variety PHY333WRF shows uniformity and stability within thelimits of environmental influence for the traits described in Table 1.Variety PHY333WRF has been self-pollinated for a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure the homozygosity and phenotypic stability necessary to use inlarge scale, commercial production. The line has been increased both byhand and sib-pollination in isolated fields with continued observationsfor uniformity. No variant traits have been observed or are expected inPHY333WRF.

Embodiments of the present disclosure also relate to one or more cottonplant parts of cotton plant PHY333WRF. Cotton plant parts include plantcells, plant protoplasts, plant cell tissue cultures from which cottonplants can be regenerated, plant DNA, plant calli, plant clumps, andplant cells that are intact in plants or parts of plants, such asembryos, ovules, pollen, stigmas, flowers, petals, seeds, bolls,gossypol glands, stems, leaves, fibers, roots, root tips, and the like.

B. Cotton Seed Designated PHY333WRF

A cotton seed is composed of three structural parts: (1) the pericarp,which is a protective outer covering (also known as bran or hull); (2)the germ (also known as an embryo); and (3) the endosperm. Anotheraspect of the present disclosure relates to one or more parts of cottonseed PHY333WRF, such as the pericarp of cotton seed PHY333WRF or thegerm and/or the endosperm of cotton seed PHY333WRF, which remain uponremoval of the pericarp and adhering remnants of the seed coat.

Cotton seed designated PHY333WRF can be provided as a substantiallyhomogenous composition of cotton seed designated PHY333WRF, that is, acomposition that consists essentially of cotton seed PHY333WRF. Such asubstantially homogenous composition of cotton seed PHY333WRF issubstantially free from significant numbers of other varietal and/orhybrid seed so that the varietal seed forms from about 90% to about 100%of the total seed. Preferably, a substantially homogenous composition ofthe varietal cotton seed contains from about 98.5%, 99%, or 99.5% toabout 100% of the varietal seed, as measured by seed grow outs. Thesubstantially homogenous composition of varietal cotton seed ofembodiments of the disclosure can be separately grown to providesubstantially homogenous populations of varietal cotton plants. However,even if a population of varietal cotton plants is present in a fieldwith other different cotton plants, such as in a commercialseed-production field of single-cross hybrid cotton planted in a ratioof 1 male pollinator row to 4 female seed-parent rows, such a populationwill still be considered to be within the scope of embodiments of thepresent disclosure.

Cotton yield is affected by the conditions to which seeds and seedlings(young plants grown from seeds) are exposed. Seeds and seedlings can beexposed to one of, or a combination of, for example, cold, drought,salt, heat, pollutants, and disease, all of which are conditions thatpotentially retard or prevent the growth of crops therefrom. Forexample, temperature extremes are typical in the United States.Furthermore, diseases evolved from pathogens and deterioration caused byfungi are potentially harmful to seeds and seedlings. Thus, it isdesirable to treat seeds by, for example, coating or impregnating theseeds with compositions that render the seeds and seedlings growntherefrom more hardy when exposed to such adverse conditions.

Accordingly, another aspect of the present disclosure relates to acoated and/or impregnated seed or cotton variety designated PHY333WRFand to coated and/or impregnated seed derived therefrom. Various agentshave been used to treat seeds to increase resistance of the plants tostressed conditions, such as cold, drought, salt, and fungi. Such agentsinclude, for example, sodium methylphenyl-pentadienate, trichloroaceticacid, polyoxyalkylene-organo-siloxane block copolymer, 5-aminolevulinicacid, salicylic acid, thiamethoxam, potassium chloride, and polyvinylalcohol and are useful alone, or in combination in embodiments of thepresent disclosure.

When pre-treating seeds in accordance with embodiments of the presentdisclosure, such as before the seeds are planted, the seeds arecontacted with the composition of interest, for example by coatingseeds, spraying seeds, soaking seeds, or a combination thereof, bymethods well known to those skilled in the art.

C. Deposit Information

Applicants have made a deposit of at least 2,500 seeds of cotton varietyPHY333WRF with the American Type Culture Collection (ATCC), Manassas,Va. 20110 USA, under ATCC Accession No. PTA-120742. The seeds depositedwith the ATCC on Dec. 2, 2013 were taken from a repository maintained byPhytogen Seed Company since before the filing date of this application.Access to the ATCC deposit will be available during the pendency of theapplication to the Commissioner of Patents and Trademarks and personsdetermined by the Commissioner to be entitled thereto upon request. Uponallowance of any claims in the application, the Applicant(s) willmaintain and will make this deposit available to the public pursuant tothe Budapest Treaty.

III. Processes of Preparing Novel Cotton Plants

A. Novel Cotton Plants Obtained From Variety PHY333WRF

Various breeding schemes can be used to produce new cotton varietiesfrom cotton variety PHY333WRF. In one method, generally referred to asthe pedigree method, PHY333WRF can be crossed with another differentcotton plant such as a second parent cotton plant variety, which eitheritself exhibits one or more selected desirable characteristic(s) orimparts selected desirable characteristic(s) to a hybrid combination.Examples of potentially desired characteristics include greater yield,better stalks, better roots, reduced time to crop maturity, better fiberquality (e.g. fineness, length, length uniformity, strength,reflectance), better storm resistance, better agronomic quality, highernutritional value, higher starch extractability or starchfermentability, resistance and/or tolerance to insecticides, herbicides,pests, heat and drought, and disease, and uniformity in germinationtimes, stand establishment, growth rate, maturity and boll size. If thetwo original parent cotton plants do not provide all the desiredcharacteristics, then other sources can be included in the breedingpopulation. Elite varieties can also be used as starting materials forbreeding or source populations from which to develop new varieties.

Thereafter, resulting seed is harvested and resulting superior progenyplants are selected and selfed or sib-mated in succeeding generations,such as for about 5 to about 7 or more generations, until a generationis produced that no longer segregates for substantially all factors forwhich the varietal parents differ, thereby providing a large number ofdistinct, pure-breeding varieties.

In another embodiment for generating new cotton varieties, generallyreferred to as backcrossing, one or more desired traits can beintroduced into parent cotton plant variety PHY333WRF (the recurrentparent) by crossing the PHY333WRF plants with another cotton plant(referred to as the donor or non-recurrent parent), which carries thegene(s) encoding the particular trait(s) of interest to produce F₁progeny plants. Both dominant and recessive alleles can be transferredby backcrossing. The donor plant can also be a varietal cotton plant,but in the broadest sense can be a member of any plant variety orpopulation cross-fertile with the recurrent parent. Next, F₁ progenyplants that have the desired trait are selected. Then, the selectedprogeny plants are crossed with PHY333WRF to produce backcross progenyplants. Thereafter, backcross progeny plants comprising the desiredtrait and the physiological and morphological characteristics of cottonvariety PHY333WRF are selected. This cycle is repeated for about one toabout eight cycles, preferably for about 3 or more times in successionto produce selected higher backcross progeny plants that comprise thedesired trait and all of the physiological and morphologicalcharacteristics of cotton variety PHY333WRF listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions. Exemplary desired trait(s) include insectresistance, cytoplasmic male sterility, enhanced fiber quality, enhancednutritional quality, herbicide resistance, yield stability, yieldenhancement, storm resistance, drought tolerance, and resistance tobacterial, fungal, nematode and viral disease. One of ordinary skill inthe art of plant breeding will appreciate that a breeder uses variousmethods to help determine which cotton plants will be selected from thesegregating populations and ultimately which varieties will be usedcommercially and will be used to develop hybrids for commercialization.In addition to the knowledge of the germplasm and other skills thebreeder uses, a part of the selection process is dependent onexperimental design coupled with the use of statistical analysis.Experimental design and statistical analysis are used to help determinewhich plants, which family of plants, and finally which varieties andhybrid combinations are significantly better or different for one ormore traits of interest. Experimental design methods are used to assesserror so that differences between two varieties or two hybrid lines canbe more accurately determined. Statistical analysis includes thecalculation of mean values, determination of the statisticalsignificance of the sources of variation, and the calculation of theappropriate variance components. Either a five or a one percentsignificance level is customarily used to determine whether a differencethat occurs for a given trait is real or due to the environment orexperimental error. One of ordinary skill in the art of plant breedingknows how to evaluate the traits of two plant varieties to determine ifthere is no significant difference between the two traits expressed bythose varieties. For example, see Fehr, Walt, Principles of CultivarDevelopment, p. 261-286 (1987), which is incorporated herein byreference in its entirety. Mean trait values can be used to determinewhether trait differences are significant, and preferably the traits aremeasured on plants grown under the same environmental conditions.

This method results in the generation of cotton plants withsubstantially all of the desired morphological and physiologicalcharacteristics of the recurrent parent and the particular transferredtrait(s) of interest. Because such cotton plants are heterozygous forloci controlling the transferred trait(s) of interest, the lastbackcross generation is subsequently selfed to provide pure breedingprogeny for the transferred trait(s).

Backcrossing can be accelerated by the use of genetic markers such asSSR, RFLP, SNP or AFLP markers to identify plants with the greatestgenetic complement from the recurrent parent.

Direct selection can be applied where a single locus acts as a dominanttrait, such as the herbicide resistance trait. For this selectionprocess, the progeny of the initial cross are sprayed with the herbicidebefore the backcrossing. The spraying eliminates any plants that do nothave the desired herbicide resistance characteristic, and only thoseplants that have the herbicide resistance gene are used in thesubsequent backcross. In some embodiments where the characteristic beingtransferred is a recessive allele, it is advisable to introduce a testof the progeny to determine if the desired characteristic has beensuccessfully transferred. The process of selection, whether direct orindirect, is then repeated for all additional backcross generations.

It will be appreciated by those having ordinary skill in the art thatbackcrossing can be combined with pedigree breeding, as where varietyPHY333WRF is crossed with another cotton plant, the resultant progenyare crossed back to variety PHY333WRF and thereafter, the resultingprogeny of this single backcross are subsequently inbred to develop newvarieties. This combination of backcrossing and pedigree breeding isuseful when recovery is desired of fewer than all of the PHY333WRFcharacteristics that will be obtained by a conventional backcross.

In an additional embodiment of the present disclosure, new cottonvarieties can be developed by a method generally referred to as haploidbreeding. In this methodology, haploid plants are generated fromdiploid, heterozygous cotton plants that result from crossing cottonplant variety PHY333WRF with another, different cotton plant. Suchhaploid cotton plants can be generated by methods known to those skilledin the art such as by culturing haploid anthers or embryos from adiploid plant. Alternately, such haploid cotton plant can be generatedby crossing the diploid heterozygous cotton plant with a cotton plantthat comprises a haploid inducing gene, which, when present in thefemale parent results in offspring with a greatly enhanced frequency ofhaploids of both maternal and paternal origin. Thereafter, homozygousdiploid plants are produced by the doubling of a set of chromosomes (IN)from a haploid plant generated by self-pollination such as through useof a doubling agent, such as colchicine, nitrous oxide gas, heattreatment and trifluralin. The technique of haploid breeding isadvantageous because no subsequent inbreeding is required to obtain ahomozygous plant from a heterozygous source. Thus, in another aspect ofthis disclosure, a new cotton plant variety is developed by a methodthat includes the steps of crossing PHY333WRF or a hybrid made withPHY333WRF with another cotton plant having a propensity to generatehaploids to produce haploid progeny plants, and selecting desirablecotton plants from the haploid progeny plants.

Embodiments of the present disclosure also relate to novel cotton plantsproduced by a method generally referred to as mutation breeding, wherebyone or more new traits can be artificially introduced into cottonvariety PHY333WRF. The goal of artificial mutagenesis is to increase therate of mutation for a desired characteristic. Mutation rates can beincreased by use of many different factors, including: temperature;long-term seed storage; tissue culture conditions; radiation, such asX-rays, Gamma rays (e.g. Cobalt-60 or Cesium-137), neutrons, (product ofnuclear fission by Uranium-235 in an atomic reactor), Beta radiation(emitted from radioisotopes such as Phosphorus-32 or Carbon-14), orultraviolet radiation (preferably from 2500 to 2900 nm); or chemicalmutagens, such as base analogues (5-bromo-uracil), related compounds(8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents(sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis andselected, the trait can then be incorporated into existing germplasm bytraditional breeding techniques. Details of mutation breeding can befound in “Principles of Cultivar Development”, Fehr, 1993 MacmillanPublishing Company, the disclosure of which is incorporated herein byreference in its entirety.

The mutagenesis treatment can be applied to various stages of plantdevelopment, including but not limited to cell cultures, embryos,microspores and shoot apices as well as to cotton seeds. By way ofexample, pollen can be mixed with a solution of 1 mL EMS and 100 mLsFisher paraffin oil (stock diluted by 1 mL and 15 mLs oil solution)every minute for the first 5 minutes and then every five minutes for 45minutes to keep the pollen suspended. Thereafter, the pollen/paraffinoil solution is brushed onto the stigmas of emasculated flower buds. Apaper soda straw is used to cover the stigma to prevent contamination.The cotton boll is picked at maturity and then resultant seeds or theplants therefrom are screened for the desired mutant trait(s).

Once new varieties are created; the next step is to determine if the newvarieties have any value. This is accomplished by techniques ofmeasuring the combining ability of the new varietal plant, as well asthe performance of the variety itself. Combining ability refers to avariety's contribution as a parent when crossed with other varieties toform hybrids. Specific combining ability (SCA) refers to the ability ofa variety to cross to another specific variety to form a hybrid. Generalcombining ability (GCA) refers to the ability of a variety to cross to awide range of varieties to form hybrids. The methodology of forminghybrids to evaluate a variety's contribution as a parent for the purposeof selecting superior varieties is interchangeably known asexperimental, top or test crossing.

B. Novel Varieties Obtained from a Hybrid Having Variety PHY333WRF as aParent

In accordance with embodiments of the present disclosure, a hybrid planthaving variety PHY333WRF as a parent is crossed with itself or anydifferent cotton plant such as a varietal cotton plant or a hybridcotton plant to develop a novel variety. For example, a hybrid cottonplant having cotton plant variety PHY333WRF as a parent can be inbred,i.e., crossed to itself or sib-pollinated, and the resulting progenyeach selfed for about 5 to about 7 or more generations, therebyproviding a set of distinct, relatively pure-breeding varieties whereineach of the varieties received all of its alleles from the hybrid cottonplant having cotton plant variety PHY333WRF as a parent. Double haploidmethods can also be used to obtain a cotton plant variety that ishomozygous at essentially every locus, wherein the cotton plant varietyreceived all of its alleles from the hybrid cotton plant having cottonplant PHY333WRF as a parent. In other embodiments, a hybrid cotton planthaving cotton plant variety PHY333WRF as a parent is crossed with adifferent cotton plant, such as any varietal cotton plant that is notvarietal cotton plant PHY333WRF, any hybrid cotton plant that does nothave PHY333WRF as a parent, another germplasm source, a haploid ormutation inducing stock, or a trait donor plant, thereby providing a setof distinct, relatively pure-breeding varieties. The resulting varietiescan then be crossed with other varieties or other cotton germplasm andthe resulting progeny analyzed for beneficial characteristics. In thisway, novel varieties conferring desirable characteristics can beidentified.

C. “Chasing Selfs”

In the event that commercial cotton hybrids are developed, both femaleand male varietal seed is occasionally found within a commercial bag ofhybrid seed. Chasing the selfs involves identifying parental varietalplants within a stand of cotton that has been grown from a bag of hybridcotton seed. Once the seed is planted, the parental plants can beidentified and selected due to their variance from the population norm,i.e., by their stature, fruiting branch structure, leaf shape, leafpubescence, fiber quality traits, or yield components relative to thehybrid plants that grow from the hybrid seed that predominates in acommercial bag of hybrid seed. By locating the parental plants,isolating them from the rest of the plants, and self-pollinating them(i.e., “chasing selfs”), a breeder can obtain a variety that isidentical to a parent used to produce the hybrid.

Accordingly, another embodiment of the present disclosure is directed toa method for producing cotton plant variety PHY333WRF comprising: (a)planting a collection of seed, such as a collection of seed comprisingseed of a hybrid, one of whose parents is cotton variety PHY333WRF, thecollection also comprising seed of the variety; (b) growing plants fromsaid collection of seed; (c) identifying parent plants; (d) controllingpollination in a manner that preserves substantial homozygosity of theparent plant; and, (e) harvesting resultant seed. Step (c) can furthercomprise identifying plants with decreased vigor, i.e., plants thatappear less robust than the other plants, or identifying plants thathave a genetic profile in accordance with the genetic profile ofPHY333WRF. Cotton plants capable of expressing substantially all of thephysiological and morphological characteristics of cotton varietyPHY333WRF include cotton plants obtained by chasing selfs from a bag ofhybrid seed.

One having skill in the art will recognize that once a breeder hasobtained cotton variety PHY333WRF by chasing selfs from a bag of hybridseed, the breeder can then produce new varietal plants such as bysib-pollinating, i.e., crossing the cotton plant PHY333WRF with anothercotton plant PHY333WRF, or by crossing the cotton plant PHY333WRF with ahybrid cotton plant obtained by growing the collection of seed.

IV. Novel Hybrid Plants

A. Novel Hybrid Seeds and Plants

In yet another aspect of the disclosure, processes are provided forproducing cotton seeds or plants, which processes generally comprisecrossing a first parent cotton plant with a second parent cotton plant,wherein at least one of the first parent cotton plant or the secondparent cotton plant is parent cotton plant variety PHY333WRF. In someembodiments of the present disclosure, the first cotton plant variety isPHY333WRF and is a female and in other embodiments the first cottonplant variety is PHY333WRF and is a male. These processes can be furtherexemplified as processes for preparing hybrid cotton seed or plants,wherein a first cotton plant variety is crossed with a second cottonplant of a different, distinct variety to provide a hybrid that has, asone of its parents, the cotton plant variety PHY333WRF. In this case, asecond variety is selected that confers desirable characteristics whenin hybrid combination with the first variety. In these processes,crossing will result in the production of seed and lint. The seed andlint production occurs regardless whether the seed and/or lint arecollected.

Any time the cotton plant variety PHY333WRF is crossed with another,different cotton variety, a first generation (F₁) cotton hybrid plant isproduced. As such, an F₁ hybrid cotton plant can be produced by crossingPHY333WRF with any second cotton plant variety. Therefore, any F₁ hybridcotton plant or cotton seed that is produced with PHY333WRF as a parentis within the scope of embodiments of the present disclosure.

When cotton plant variety PHY333WRF is crossed with another cotton plantvariety to yield a hybrid, the original variety can serve as either thematernal or paternal plant with, basically, the same characteristics inthe hybrids. Occasionally, maternally inherited characteristics canexpress differently depending on the decision of which parent to use asthe female. However, often one of the parental plants is preferred asthe maternal plant because of increased seed and/or lint yield andpreferred production characteristics, such as optimal seed size andquality or ease of boll or lint removal. Particularly in very hotclimates, such as in the Southwest USA, pollen can be shed better by oneplant, making that plant the preferred male parent. It is generallypreferable to use PHY333WRF as the male parent.

In embodiments of the present disclosure, the first step of “crossing”the first and the second parent cotton plants comprises planting,preferably in pollinating proximity, seeds of a first cotton plantvariety and a second, distinct cotton plant variety. As discussedherein, the seeds of the first cotton plant variety and/or the secondcotton plant variety can be treated with compositions that render theseeds and seedlings grown therefrom more hardy when exposed to adverseconditions.

A further step comprises cultivating or growing the seeds of the firstand second parent cotton plants into plants that bear flowers. If theparental plants differ in timing of sexual maturity, techniques can beemployed to obtain an appropriate nick, i.e., to ensure the availabilityof pollen from the parent cotton plant designated the male during thetime at which stigmas on the parent cotton plant designated the femaleare receptive to the pollen. Methods that can be employed to obtain thedesired nick include delaying the flowering of the faster maturingplant, such as, but not limited to, delaying the planting of the fastermaturing seed, cutting or burning the top leaves of the faster maturingplant (without killing the plant) or speeding up the flowering of theslower maturing plant, such as by covering the slower maturing plantwith film designed to speed germination and growth.

In a preferred embodiment, the cotton plants are treated with one ormore agricultural chemicals as considered appropriate by the grower.

A subsequent step comprises preventing self-pollination orsib-pollination of the plants, i.e., preventing the stigmas of a plantfrom being fertilized by any plant of the same variety, including thesame plant. This is preferably done in large scale production bycontrolling the male fertility, e.g., treating the flowers so as toprevent pollen production or alternatively, using as the female parent amale sterile plant of the first or second parent cotton plant (i.e.,treating or manipulating the flowers so as to prevent pollen production,to produce an emasculated parent cotton plant, or using as a female acytoplasmic male sterile version of the cotton plant). This control canalso be accomplished in small scale production by physical removal ofthe staminal column of individual flowers before anthesis to provideeffective control of unwanted self-pollination or sib-pollination.

Yet another step comprises allowing cross-pollination to occur betweenthe first and second parent cotton plants. When the plants are not inpollinating proximity, this is done by either collecting ripe,undehisced anthers from a flower on the pollen parent with a shortsection of a soda straw during the same evening of the emasculations, orcollecting whole, freshly dehisced flowers during the next morning afterthe emasculations. The soda straw containing the ripe anthers is thenslipped over the stigma of an emasculated flower. Finally, bracts arewired around the soda straw, holding it in place over the style, thusprotecting the stigma from foreign pollen. If a whole flower from themale parent is used, the petals are folded down and the staminal columnis rubbed onto the emasculated stigma. In small-scale production, seedsof hybrid cotton are commercially produced by hand emasculation andpollination, or by hand pollination of genetic male-sterile cotton. Inlarge scale production, seed of hybrid cotton are commercially producedby using various bees and other insect pollinators to cross pollinategenetic or cytoplasmic male-sterile cotton, or cotton that has beentreated with a chemical that results in male sterility.

A further process comprises harvesting the seeds and/or lint, near or atmaturity, from the bolls of the plants that received the pollen. In aparticular embodiment, seed and/or lint are harvested from the femaleparent plant, and when desired, the harvested seed can be grown toproduce a first generation (F₁) hybrid cotton plant.

Yet another process comprises ginning the seed cotton to separate theseed from the marketable lint and delinting the “fuzzy” seed to removethe short “linters” that remain attached after ginning. The seeds arefurther conditioned and treated with chemicals such as fungicides andinsecticides prior to being packaged for sale to growers for theproduction of lint and seed. As with varietal seed, in some embodimentsit is desirable to treat hybrid seeds with compositions that render theseeds and seedlings grown therefrom more hardy when exposed to adverseconditions. The resulting hybrid seed is sold to growers for theproduction of seed and lint and not generally for breeding.

Further embodiments of the present disclosure relate to a hybrid cottonplant produced by growing the harvested seeds produced on themale-sterile plant, as well as seed produced by the hybrid cotton plant.

A single cross hybrid is produced when two different parent cotton plantvarieties are crossed to produce first generation F₁ hybrid progeny.Generally, each parent cotton plant variety has a genotype thatcomplements the genotype of the other parent variety. Typically, the F₁progeny are more vigorous than the respective parent cotton plantvarieties. This hybrid vigor, or heterosis, is manifested in manypolygenic traits, including markedly improved yields and improvedfruiting, roots, uniformity and insect and disease resistance. It is forthis reason that single cross F₁ hybrids are generally the mostsought-after hybrid. A three-way, or modified single-cross hybrid isproduced from three varieties where two of the varieties are crossed(A×B) and then the resulting F₁ hybrid is crossed with the third variety(A×B)×C, as where a modified female is used in the cross. A modifiedfemale provides an advantage of improved seed/lint parent yield whereasa modified male improves pollen flow. A double cross hybrid is producedfrom four varieties crossed in pairs (A×B and C×D), thereby resulting intwo F₁ hybrids that are crossed again. Double cross hybrids are morecommon in countries wherein less demand exists for higher yieldingsingle cross hybrids. Synthetic populations or crosses are developed bycrossing two or more varieties (or hybrids, or germplasm sources)together and then employing one of many possible techniques to randommate the progeny. Random mating the progeny is any process used by plantbreeders to make a series of crosses that will create a new germplasmpool from which new breeding germplasm can be derived. Since crosspollination of male sterile cotton plants by hand or by various insectsis generally very inefficient, F₁ hybrid seed is generally too expensiveto produce on a large scale. Consequently, in some embodiments the F₂seed harvested from F₁ hybrids retains suitable heterosis to be aneconomically viable option to pure-line varieties.

The utility of the cotton plant variety PHY333WRF also extends tocrosses with species other than the hirsutum species, such asbarbadense. Commonly, suitable species will be of the family Malvaceae,and especially of the genera Gossypium.

B. Cotton Varietal Comparison

As mentioned above, experimental strains are progressively eliminatedfollowing detailed evaluations of their phenotype, including formalcomparisons with other commercially successful varieties. Researchsmall-plot trials and commercial strip trials are used to compare thephenotypes of varieties grown in as many environments as possible. Theyare performed in many environments to assess overall performance of thenew varieties and to select optimum growing conditions. Because thecotton strains and varieties are grown in close proximity, differentialeffects of environmental factors that affect gene expression, such asmoisture, temperature, sunlight, and pests, are minimized. For adecision to be made to advance a strain, it is not necessary that thestrain be better than all other varieties. Rather, significantimprovements must be shown in at least some traits that will createvalue for some applications or markets. Some experimental strains areeliminated, despite being similarly competitive relative to the currentcommercial varieties, because the cost to bring a new variety to marketrequires a new product to be a significant improvement over the existingproduct offering. Such varieties can also be licensed to other partieswho have a need in their commercial product portfolio.

PHY333WRF was evaluated for lint yield at 50 locations with 3 or 4replications per location in 2011 and 2012. The test locations were nearBelle Mina, Ala.; Fairhope, Ala.; Headland, Ala.; Shorter, Ala.; JuddHill, Ark.; Leachville, Ark.; Marianna, Ark.; Portland, Ark.;Bainbridge, Ga.; Chula, Ga.; Moultrie, Ga.; Midville, Ga.; Unadilla,Ga.; Willacoochee, Ga.; Crowville, La.; St. Joseph, La.; Clarkton, Mo.;Vanduser, Mo.; Portageville, Mo.; Leland, Miss.; Clarksdale, Miss.;Rocky Mount, N.C.; Washington, N.C.; Elko, S.C.; Florence, S.C., Milan,Tenn.; Groom, Tex., Halfway, Tex.; Lubbock, Tex.; San Angelo, Tex.; andSuffolk, Va. Compared to PHY367WRF and PHY375WRF, leading market Uplandvarieties adapted to the Delta and Eastern cotton-growing regions,PHY333WRF had significantly greater lint yield over all locations (Table2). In addition, the maturity of PHY333WRF was earlier than bothPHY367WRF and PHY499WRF, as indicated by the percent of open bolls(Table 2).

TABLE 2 Comparison of Lint Yield and Open Bolls¹ Between PHY333WRF andSimilarly Adapted Cotton Cultivars Lint Yield Open Bolls Cultivar(lbs/acre)² (%)³ PHY333WRF 1476 a  76.8 a  DP0912B2RF 1248 bc notavailable DP1028B2RF 1245 bc not available DP1050B2RF 1254 bc notavailable PHY367WRF 1196 c  66.4 abc PHY375WRF 1246 bc not availablePHY499WRF 1421 a  63.7 bc  Means followed by the same letter do notsignificantly differ; calculated using Tukey's W procedure at 95%¹Higher percentage open bolls indicates earlier maturity at harvest.²Means across 50 environments. ³Means across 4 environments.

PHY333WRF appears stable and uniform in isolated field seed productionand field-trial evaluations, and no off-type plants have been exhibited.This line has exhibited commercial value in field evaluations and iswell adapted to the Mississippi Delta and Eastern United Statesfull-season production regions. It will be of value to cotton producerswho desire a cotton variety that has insect resistance from Bt genes inaddition to herbicide resistance.

V. Novel PHY333WRF-Derived Plants

All plants produced using cotton plant variety PHY333WRF as a parent arewithin the scope of embodiments of this disclosure, including plantsderived from cotton plant variety PHY333WRF. This includes plantsessentially derived from variety PHY333WRF, where the term “essentiallyderived variety” has the meaning ascribed to such term in 7 U.S.C.§2104(a)(3), also known as Section 2104(a)(3) of the Plant VarietyProtection Act, which section is hereby incorporated by reference in itsentirety. This also includes a progeny plant and parts thereof with atleast one ancestor that is cotton plant variety PHY333WRF, and morespecifically, where the pedigree of this progeny includes 1, 2, 3, 4,and/or 5 or cross pollinations to cotton plant PHY333WRF, or a plantthat has PHY333WRF as a progenitor. All breeders of ordinary skill inthe art maintain pedigree records of their breeding programs. Thesepedigree records contain a detailed description of the breeding process,including a listing of all parental lines used in the breeding processand information on how such line was used. Thus, a breeder will know ifPHY333WRF were used in the development of a progeny line, and will alsoknow how many breeding crosses to a line other than PHY333WRF were madein the development of any progeny line. A progeny line so developed canthen be used in crosses with other, different, cotton varieties toproduce first generation F1 cotton hybrid seeds and plants with superiorcharacteristics.

Accordingly, another aspect of the present disclosure relates to methodsfor producing a PHY333WRF-derived cotton plant. Embodiments of suchmethods for producing a PHY333WRF-derived cotton plant comprise: (a)crossing cotton plant PHY333WRF with a second cotton plant to yieldprogeny cotton seed; and (b) growing the progeny cotton seed (underplant growth conditions) to yield the PHY333WRF-derived cotton plant.Such methods can further comprise the steps of: (c) crossing thePHY333WRF-derived cotton plant with itself or another cotton plant toyield additional PHY333WRF-derived progeny cotton seed; (d) growing theprogeny cotton seed of step (b) (under plant growing conditions) toyield additional PHY333WRF-derived cotton plants; and (e) repeating thecrossing and growing steps of (c) and (d) from 0 to 7 times to generatefurther PHY333WRF-derived cotton plants. Still further, this cancomprise utilizing methods of semigamy and other haploid breeding andplant tissue culture methods to derive progeny of the PHY333WRF-derivedcotton plant.

VI. Tissue Cultures and In Vitro Regeneration of Cotton Plants

As is well known in this art, tissue culture of cotton can be used forthe in vitro regeneration of a cotton plant. Accordingly, furtheraspects of the disclosure relate to tissue cultures of the cotton plantvariety designated PHY333WRF, to tissue cultures of hybrid and derivedcotton plants obtained from PHY333WRF, to plants obtained from suchtissue cultures and to the use of tissue culture methodology in plantbreeding. The term “tissue culture” includes a composition comprisingisolated cells of the same type, isolated cells of different types, or acollection of such cells organized into parts of a plant. Exemplarytissue cultures are protoplasts, calli and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,petals, seeds, bolls, gossypol glands, stems, leaves, fibers, roots,root tips, and the like. In a preferred embodiment, the tissue culturecomprises embryos, protoplasts, meristematic cells, pollen, leaves oranthers derived from immature tissues of these plant parts.

A. Cotyledon Culture

To obtain plant tissue for callus culture initiation, seeds areharvested from a wild type cotton plant (generally GC510 or Coker310genotype). Initially, seeds are surface sterilized by a triple rinsewith 70% ethanol for 1 minute each, a thorough rinse with sterile water,followed by a wash in 30% commercial bleach (0.1% sodium hypochlorite)for about 20 minutes.

Seeds are rinsed in sterile distilled water, and seeds are placed on thesurface of germination media (LS salts (10×), 3% sucrose, modified B5vitamins (1000×), at pH 5.8) for the production of sterile plantlets. Atapproximately, 7-10 days post plating, plantlets will have emerged fromthe seeds. The “first true leaves” are the cotyledons. Generally, tissueculture media contains amino acids, salts, sugars, hormones, andvitamins. The proportion of one ingredient versus another depends on theapplication (e.g., need for rooting versus shoot elongation). At day7-10, the cotyledons are of sufficient size for experimental use. Thecotyledons are cut into 1 mm square pieces and plated on callusinduction media (100 mL/L LS salts (10×), 3% glucose, 1 mL/L modified B5vitamins (1000×), 1 ml/L 1 mM kinetin, 1 ml/L 1 mM 2,4-D, 8 g/L nobleagar, pH 5.8). The cotyledon segment is placed on the media in theabaxial side down orientation. After three weeks on the callus inductionmedia, callus forms around the cut edges of the segment; the callus isremoved from the edges using a scalpel. The “callus” is a loosecollection or mass of undifferentiated cells, which can be yellow-greenin color. Some lines are prone to phenolic production (browning), whichcan affect growth. The callus is maintained on the initiation media fornine weeks, with subculture to fresh media every three weeks. If thesegments undergo transformation, they are co-cultured with Agrobacteriumin callus induction media for 3 days and then transferred to callusinduction media supplemented with carbenicillin, which is an antibioticto kill the Agrobacterium (2 ml/L), and glufosinate-ammonium (0.5 ml/L),which is the selective agent that allows growth of only those cells thatcontain a transgene (PAT).

At week nine, the callus is transferred to growth media (100 ml/L LSsalts, 3% glucose, 1 ml/L B5 vitamins, 4.6 ml/L kinetin, 10.7 mL/L NAA,8 g/L noble agar, pH 5.8, and, if Agrobacterium infection was used totransfer the PAT gene, carbenicillin (0.4 ml/L) and glufosinate ammonium(0.3 ml/L)). The callus will remain on this media for 3 weeks, to allowfor increased growth before going to embryogenic callus induction media.Once sufficient callus is present, the tissue is placed on embryogenicinduction media (1 pkg DKW salts, 10 ml/L myo-inositol, 1 ml/L B5vitamins, 2% glucose, 8 g/L noble agar, pH 5.8). The time for a line toproduce embryogenic callus varies from two to six months; during whichtime the callus remains on the same plate of media. Stress can assist ininducing cotton callus to become embryogenic.

Regeneration begins with embryogenic callus. Embryogenic callus ismaintained on the embryogenic callus induction media, with two weeksubcultures to fresh media. Microscope use is preferred for theisolation and transfer of embryogenic callus to ensure the desiredmorphology is taken from the plates. The desired morphology has agranular appearance, yellow-green in color. The embryogenic callus willgive rise to embryos, which can look like small footballs and have agreen color. The embryos mature on the embryogenic callus inductionmedia. It can take three to nine weeks for the embryos to mature orelongate; transfers are carried out at three-week intervals. At themature or elongated stage the embryos are transferred to basal mediathat will improve shoot (1 pkg DKW salts, 10 mL/L myo-inositol, 1 mL/Lmodified B5 vitamins, 3% sucrose, 0.5 mL/L kinetin, 8 g/L noble agar, pH5.8) or root development (0.5 pkg DKW salts, 5 mL/L myo-inositol, 0.5mL/L modified B5 vitamins, 1% sucrose, 8 g/L noble agar, pH 5.8).

When secondary roots have formed and the shoot is 1 to 2 inches highwith 2 good leaves, the cotton plant is ready for soil. Plantlets arefirst placed in a Conviron in small pots with a humidi-dome to assistwith plant hardening, since cotton plants can be quite fragile. Thenplants are later transferred to large pots in the greenhouse. Mostcotton plants are allowed to self-pollinate and these flowers are taggedwith one color, while others can be crossed with an elite variety andtagged separately.

B. Additional Tissue Cultures and Regeneration

Other methods for preparing and maintaining plant tissue cultures arewell known in the art. By way of example, reference may be had toKomatsuda, T. et al., Crop Sci. 31:333-337 (1991); Stephens, P. A., etal., Theor. Appl. Genet. 82:633-635 (1991); Komatsuda, T. et al., PlantCell, Tissue and Organ Culture, 28:103-113 (1992); Dhir, S. et al.,Plant Cell Reports 11:285-289 (1992); Pandey, P. et al., Japan J. Breed.42:1-5 (1992); and Shetty, K., et al., Plant Science 81:245-251 (1992);as well as U.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins etal., and U.S. Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch et al.Thus, another aspect of this disclosure relates to cells that upongrowth and differentiation produce cotton plants having thephysiological and morphological characteristics of the present cottonvariety.

VII. Male Sterility

Methods for controlling male fertility in cotton plants offer theopportunity for improved plant breeding, particularly for thedevelopment of cotton hybrids that require the implementation of a malesterility system to prevent the varietal parent plants fromself-pollination.

Accordingly, another aspect of the present disclosure relates tomale-sterile varietal cotton plants designated PHY333WRF and theproduction of hybrid cotton seed using a male sterility system with suchvarietal female parent plants that are male sterile. If cotton varietyPHY333WRF is employed as the female parent, PHY333WRF can be renderedmale-sterile by, for example, removing the stamens of PHY333WRF parentalplants manually. By way of example, alternate strips of two cottonvarieties can be planted in a field followed by manual emasculation.Provided that the female variety is sufficiently isolated from foreigncotton pollen sources, the stigma of the emasculated variety will befertilized only from the other male variety either manually or by insectpollinator vectors, and the resulting seed will therefore be hybridseed.

The laborious and occasionally unreliable manual emasculation processcan be minimized by using cytoplasmic male-sterile (CMS) varieties.Plants of a CMS variety are male sterile as a result of the influence ofcytoplasmic factors, rather than those of the nuclear genome. Thus, thischaracteristic is inherited exclusively through the female parent incotton plants, since CMS plants are fertilized with pollen from anothervariety that is not male-sterile. In some embodiments, pollen from thesecond variety contributes genes that make the hybrid plantsmale-fertile. Seed from emasculated fertile cotton and CMS produced seedof the same hybrid can be blended to insure that adequate pollen loadsare available for fertilization when the hybrid plants are grown.Conventional backcrossing methods can be used to introgress the CMStrait into variety PHY333WRF.

Alternatively, haploid breeding methods can also be employed to convertvariety PHY333WRF to CMS sterility. Haploids are plants that containonly one-half of the chromosome number present in diploid somatic cells,which are cells other than haploid cells, such as those found in thegerm. There are a few stocks or genetic systems in cotton that are knownto generate haploids spontaneously.

Manual emasculation can also be avoided by the use of chemically inducedmale sterility in the production of hybrid cotton seed. Chemicals thatinduce male sterility include gametocides, pollen suppressants, andchemical hybridizing agents. The general procedure is to use a foliarspray before flowering, which inhibits production of viable pollen, butdoes not injure the pistillate reproductive organs or affect seeddevelopment. If the treatment is successful and all of the pollen iskilled, self-pollination will not occur in the treated plants, but theflowers will set seed freely from cross-pollination. In such a case, theparent plants used as the male can either not be treated with thechemical agent or can include a genetic factor that causes resistance tothe sterilizing effects of the chemical agent. The use of chemicallyinduced male sterility affects fertility in the plants only for thegrowing season in which the gametocide is applied.

The presence of a male-fertility restorer gene results in the productionof a fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the cotton plant is used, e.g.,for silage, but in most cases, the seeds will be deemed the mostvaluable portion of the crop, so fertility of the hybrids in these cropsmust be restored. Therefore, one aspect of the present disclosurerelates to cotton variety PHY333WRF comprising a single gene capable ofrestoring male fertility in an otherwise male-sterile variety or hybridplant. Examples of male-sterility genes and corresponding restorers thatcan be employed within the scope of embodiments of the disclosure arewell known to those of skill in the art of plant breeding and aredisclosed in, for example, U.S. Pat. Nos. 5,530,191, 5,689,041,5,741,684, and 5,684,242, the disclosures of which are each specificallyincorporated herein by reference in their entirety.

VIII. Cotton Transformation

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and to expressforeign genes, or additional, or modified versions of native orendogenous genes (perhaps driven by different promoters) to alter thetraits of a plant in a specific manner. Such foreign, additional and/ormodified genes are referred to herein collectively as “transgenes.” Thepresent disclosure, in particular embodiments, also relates totransformed versions of the claimed cotton variety PHY333WRF containingone or more transgenes.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement. The expression vector can contain one or more such operablylinked gene/regulatory element combinations. The vector(s) can be in theform of a plasmid, and can be used, alone or in combination with otherplasmids, to develop transformed cotton plants, using transformationmethods as described below to incorporate transgenes into the geneticmaterial of the cotton plant(s).

A. Expression Vectors for Cotton Transformation/Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element that allows transformed cells containing themarker to be either recovered by negative selection, i.e., inhibitinggrowth of cells that do not contain the selectable marker gene, or bypositive selection, i.e., screening for the product encoded by thegenetic marker. Many commonly used selectable marker genes for planttransformation are well known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent that can be an antibiotic or a herbicide, orgenes that encode an altered target that is insensitive to theinhibitor. A few positive selection methods are also known in the art.One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from a bacterialsource, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A. 80: 4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene that confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol. 5: 299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferaseand the bleomycin resistance determinant. Hayford et al., Plant Physiol.86: 1216 (1988), Jones et al., Mol. Gen. Genet. 210: 86 (1987), Svab etal., Plant Mol. Biol. 14: 197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317: 741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618(1990) and Stalker et al., Science 242: 419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67(1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant CellRep. 8: 643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, and luciferase. Jefferson, R. A., Plant Mol. Biol. Rep.5: 387 (1987), Teeri et al., EMBO J. 8: 343 (1989), Koncz et al., Proc.Natl. Acad. Sci. U.S.A. 84: 131 (1987). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247: 449(1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes Publication 2908, Imagene Green™, p. 1-4 (1983) and Naleway etal., J. Cell Biol. 115: 151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263: 802 (1994). GFP and mutants of GFPcan be used as screenable markers.

B. Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in certain tissues arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example vascular cells in roots or leaves. An “inducible” promoteris a promoter that is under environmental control or is induced inresponse to chemical or hormonal stimuli. Examples of environmentalconditions that can effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Examples of chemicalsthat induce expression include salicylic acid and ABA. Tissue-specific,tissue-preferred, cell type specific, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter that is active under most environmental conditions and in allcells.

1. Inducible Promoters

An inducible promoter is operably linked to a gene for expression incotton. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence that is operably linkedto a gene for expression in cotton. With an inducible promoter the rateof transcription increases in response to an inducing agent. Anyinducible promoter can be used in embodiments of the instant disclosure.A particularly preferred inducible promoter is a promoter that respondsto an inducing agent to which plants do not normally respond. Anexemplary inducible promoter is the inducible promoter from a steroidhormone gene, the transcriptional activity of which is induced by aglucocorticosteroid hormone.

2. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression incotton or is operably linked to a nucleotide sequence encoding a signalsequence that is operably linked to a gene for expression in cotton.Many different constitutive promoters can be used in embodiments of thepresent disclosure. Exemplary constitutive promoters include, but arenot limited to, the promoters from plant viruses such as the 35Spromoter from CaMV and the promoters from such genes as rice actin,maize ubiquitin, and corn H3 histone. Also, the ALS promoter, anXbaI/NcoI fragment 5′ to the Brassica napus ALS3 structural gene (or anucleotide sequence that has substantial sequence similarity to theXbaI/NcoI fragment) represents a particularly useful constitutivepromoter.

3. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin cotton. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence that is operablylinked to a gene for expression in cotton. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue. Any tissue-specific or tissue-preferred promoter canbe utilized in embodiments of the instant disclosure. Exemplarytissue-specific or tissue-preferred promoters include, but are notlimited to, a seed-preferred promoter such as that from the phaseolingene; a leaf-specific and light-induced promoter such as that from cabor rubisco; an anther-specific promoter such as that from LAT52; apollen specific promoter such as that from Zm13; or amicrospore-preferred promoter such as that from apg.

C. Signal Sequences For Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion, or for secretion into the apoplast, is accomplished byoperably linking the nucleotide sequence encoding a signal sequence tothe 5′ and/or 3′ region of a gene encoding the protein of interest.Targeting sequences at the 5′ and/or 3′ end of the structural gene candetermine, during protein synthesis and processing, where the encodedprotein is ultimately compartmentalized. The presence of a signalsequence directs a polypeptide to either an intracellular organelle orsubcellular compartment or for secretion to the apoplast. Use of anysignal sequence known in the art is contemplated for use in embodimentsof the present disclosure.

D. Foreign Protein Genes and Agronomic Genes

Using transgenic plants of embodiments of the present disclosure, aforeign protein can be produced in commercial quantities. Thus,techniques for the selection and propagation of transformed plants,which are well understood in the art, yield a plurality of transgenicplants, which are harvested in a conventional manner, and a foreignprotein then can be extracted from a tissue of interest or from totalbiomass. Protein extraction from plant biomass can be accomplished byknown methods.

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is cotton. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, for example via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, and Simple Sequence Repeats (SSR), in a manner that identifiesthe approximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Glick and Thompson, Methodsin Plant Molecular Biology and Biotechnology 269-284 (CRC Press, BocaRaton, 1993). Map information concerning chromosomal location is usefulfor proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. In particular embodiments, mapcomparisons can involve, for example, hybridizations, RFLP, PCR, SSR andsequencing, all of which are conventional techniques.

Likewise, in accordance with embodiments of the present disclosure,agronomic genes can be expressed in transformed plants. Moreparticularly, plants can be genetically engineered to express variousphenotypes of agronomic interest. Exemplary genes implicated in thisregard include, but are not limited to:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

(a) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones et al., Science 266: 789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin et al., Science 262: 1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos et al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae).

(b) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection (Rockville,Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

(c) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

(d) A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by referencein their entirety. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

(e) An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793(1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huubet al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci.Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).

(f) An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(g) An insect-specific peptide or neuropeptide that, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm 163: 1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

(h) An insect-specific venom produced in nature by a snake, a wasp, etc.

For example, see Pang et al., Gene 116: 165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

(i) An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(j) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule, forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, or a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules that containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

(k) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a corn calmodulin cDNA clone.

(l) A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin that inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference in theirentirety.

(m) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(n) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(o) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut will inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Intl. Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland (Edinburgh, Scotland,1994) (enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

(p) A virus-specific antibody. See, for example, Tavladoraki et al,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonate. See Lamb et al., Bio/Technology10: 1436 (1992). The cloning and characterization of a gene that encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2: 367 (1992).

(r) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to a Herbicide, for Example:

(a) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Mild et al., Theor. Appl. Genet. 80: 449(1990), respectively.

(b) Phosphono compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyltransferase (bar) genes), and pyridinoxy or phenoxypropionic acids andcyclohexones (ACCase inhibitor-encoding genes). European patentapplication No. 0 333 033 to Kumada et al. and U.S. Pat. No. 4,975,374to Goodman et al. disclose nucleotide sequences of glutamine synthetasegenes that confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al.Furthermore, De Greef et al., Bio/Technology 7: 61 (1989), describe theproduction of transgenic plants that express chimeric bar genes codingfor phospinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxypropionic acids and cyclohexones, suchas sethoxydim and haloxyfop, are the Acc1-81, Acc1-S2 and Acc1-S3 genesdescribed by Marshall et al., Theor. Appl. Genet. 83: 435 (1992).

(c) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

(d) 2,4-D and other phenoxy acid herbicides and pyridoxyloxyacetateherbicides such as fluroxypyr. See, for example, U.S. Pat. No. 8,283,522to Wright et al., which discloses the nucleotide sequence of a geneencoding an enzyme capable of degrading these herbicides.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

(a) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89: 2624 (1992).

(b) Decreased phytate content:

(i) Introduction of a phytase-encoding gene will enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(ii) A gene can be introduced that reduces phytate content. In cotton,this, for example, can be accomplished by cloning and then reintroducingDNA associated with the single allele that is responsible for cottonmutants characterized by low levels of phytic acid. See Raboy et al.,Maydica 35: 383 (1990).

(iii) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtillus levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α-amylase), Elliot et al., Plant Molec.Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes),Sogaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102: 1045 (1993) (corn endosperm starch branching enzyme II).

E. Methods for Cotton Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, inc., Boca Raton,1993) pages 89-119.

1. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria that genetically transformplant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant. Sci. 10: 1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Mild et al., supra, and Moloney et al., Plant Cell Reports 8: 238(1989). See also U.S. Pat. No. 5,591,616, issued Jan. 7, 1997.

2. Direct Gene Transfer

Several methods of plant transformation, collectively referred to asdirect gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm (See e.g., U.S. Pat. No.5,550,318; U.S. Pat. No. 5,736,369; U.S. Pat. No. 5,538,880; and PCTPublication WO 95/06128). The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s, which is sufficient to penetrate plant cellwalls and membranes. Sanford et al, Part. Sci. Technol. 5: 27 (1987),Sanford, J. C., Trends Biotech. 6: 299 (1988), Klein et al.,Bio/Technology 6: 559-563 (1988), Sanford, J. C., Physiol. Plant 79: 206(1990), Klein et al., Biotechnology 10: 268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Christouet al., Proc. Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199: 161 (1985) and Draper et al., Plant Cell Physiol. 23: 451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. U.S. Pat. No. 5,384,253 and Donn et al; in Abstracts ofVIIth International Congress on Plant Cell and Tissue Culture IAPTC,A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4: 1495-1505 (1992) andSpencer et al., Plant Mol. Biol. 24: 51-61 (1994).

Other methods that have been described for the genetic transformation ofcotton include electrotransformation (U.S. Pat. No. 5,371,003) andsilicon carbide fiber-mediated transformation (U.S. Pat. No. 5,302,532and U.S. Pat. No. 5,464,765).

Following transformation of cotton target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation are typically used forproducing transgenic cotton varieties. Transgenic cotton varieties canthen be crossed, with another (non-transformed or transformed) cottonvariety, to produce a transgenic hybrid cotton plant. Alternatively, agenetic trait that has been engineered into a particular cotton varietyusing the foregoing transformation techniques can be moved into anotherline using traditional backcrossing techniques that are well known inthe plant breeding arts. For example, a backcrossing approach can beused to move an engineered trait from a public, non-elite line into anelite line, or from a hybrid cotton plant containing a foreign gene inits genome into a line or lines that do not contain that gene.

IX. Genetic Complements

In addition to phenotypic observations, a plant can also be described byits genotype. The genotype of a plant can be described through a geneticmarker profile that can identify plants of the same variety, a relatedvariety or be used to determine or to validate a pedigree. Geneticmarker profiles can be obtained by techniques such as RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), SimpleSequence Repeats (SSRs), which are also referred to as Microsatellites,and Single Nucleotide Polymorphisms (SNPs), Isozyme Electrophoresis andIsoelectric Focusing.

Particular markers used for these purposes are not limited to the set ofmarkers disclosed herewithin, but are envisioned to include any type ofgenetically stable marker and marker profile that provides a way ofdistinguishing varieties. In addition to being used for identificationof cotton varieties, a hybrid produced through the use of PHY333WRF, andidentification or verification of the pedigree of progeny plantsproduced through the use of PHY333WRF, the genetic marker profile isalso useful in breeding and developing backcross conversions.

Methods of generating genetic marker profiles using SSR polymorphismsare well known in the art. SSRs are genetic markers based onpolymorphisms in repeated nucleotide sequences, such as microsatellites.The phrase “simple sequence repeats” or “SSR” refers to di-, tri- ortetra-nucleotide repeats within a genome. The repeat region can vary inlength between genotypes while the DNA flanking the repeat is conserved,such that the primers will work in a plurality of genotypes. Apolymorphism between two genotypes represents repeats of differentlengths between the two flanking conserved DNA sequences. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that, in some embodiments, multiplealleles are present. Another advantage of this type of marker is that,through use of flanking primers, detection of SSRs can be achieved, forexample, by the polymerase chain reaction (PCR). The PCR detection isdone by the use of two oligonucleotide primers flanking the polymorphicsegment of repetitive DNA followed by DNA amplification. This stepinvolves repeated cycles of heat denaturation of the DNA followed byannealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase.Size separation of DNA fragments on agarose or polyacrylamide gelsfollowing amplification comprises the major part of the methodology.

DNA isolation and amplification can be performed in certain embodimentsof the present disclosure as follows. DNA can be extracted from plantleaf tissue using DNeasy 96 Plant Kit from Qiagen, Inc. (Valencia,Calif., U.S.A.) following an optimized September 2002 manufacturer'sprotocol. PCR amplifications are performed using a Qiagen HotStar™ Taqmaster mix in an 8 μl reaction format as follows: 2 μl DNA (5 ng/μL+6 μLof master mix). The PCR conditions are as follows: 12 mins. at 95° C.,40 cycles of 5 seconds at 94° C., 15 seconds at 55° C., 30 seconds at72° C., 30 mins at 72° C., followed by cooling to 4° C. Followingisolation and amplification, markers can be scored by gelelectrophoresis of the amplification products. Scoring of markergenotype is based on the size of the amplified fragment as measured bymolecular weight (MW) rounded to the nearest integer. Multiple samples,comprising fluorescently labeled DNA fragments, can be processed in anABI 3700 capillary-based machine and precise allele sizing and locusgenotyping can be done by running GeneScan and Genotyper software (PEApplied Biosystems, Foster City, Calif.). When comparing varieties, itis preferable that all SSR profiles be performed in the same lab. An SSRservice is available to the public on a contractual basis by Paragen,Research Triangle Park, N.C. (formerly Celera AgGen of Davis, Calif.).

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this disclosure pertains. All such publications, patents andpatent applications are incorporated by reference herein to the sameextent as if each was specifically and individually indicated to beincorporated by reference herein.

The foregoing invention has been described in some detail by way ofillustration and example for purposes of clarity and understanding.However, it will be appreciated by those having ordinary skill in theart that certain changes and modifications such as single genemodifications and mutations, somoclonal variants, variant individualsselected from large populations of the plants of the instant variety andthe like can be practiced within the scope of the embodiments of theinvention, as limited only by the scope of the appended claims, withoutdeparting from the true concept, spirit, and scope of the invention.

What is claimed is:
 1. A seed of cotton variety designated PHY333WRF, ora part thereof, representative seed of the variety having been depositedunder ATCC Accession No. PTA-120742 on Dec. 2,
 2013. 2. A part of theseed of claim 1 selected from the group consisting of hull (seed coat),germ and endosperm.
 3. The seed of claim 1, further comprising acoating.
 4. A substantially homogenous composition of the cotton seed ofclaim
 1. 5. A method for producing a seed of a cotton plant, comprising:(a) planting the seed of claim 1 in proximity to itself or to differentseed from a same variety; (b) growing plants from the seed underpollinating conditions; and, (c) harvesting resultant seed.
 6. A cottonseed produced by the method of claim
 5. 7. The method of claim 5,further comprising pre-treating the seed of claim 1 before performingstep (a).
 8. The method of claim 5, further comprising treating thegrowing plants or soil surrounding the growing plants with anagricultural chemical.
 9. A cotton plant produced by growing the seed ofclaim
 1. 10. A part of the cotton plant of claim 9, selected from thegroup consisting of an intact plant cell, a plant protoplast, embryos,pollen, flowers, seeds, staples, linters, fibers, pods, gossypol glands,leaves, bolls, stems, roots, root tips, and anthers.
 11. Fibers of theplant of claim
 9. 12. Staples of the plant of claim
 9. 13. A cottonplant, or a part thereof, having all the physiological and morphologicalcharacteristics of the cotton plant of claim
 9. 14. A substantiallyhomogenous population of cotton plants of claim
 9. 15. The substantiallyhomogenous population of cotton plants of claim 14, wherein thepopulation is present in a field and the field further comprises other,different cotton plants.
 16. A method for producing a cotton plant,comprising: (a) crossing cotton variety plant PHY333WRF, representativeseed of the variety having been deposited under ATCC Accession No.PTA-120742 on Dec. 2, 2013, with another different cotton plant to yieldprogeny cotton seed.
 17. The method of claim 16, wherein the other,different cotton plant is a cotton variety.
 18. The method of claim 16,further comprising: (b) growing the progeny cotton seed from step (a)under self-pollinating or sib-pollinating conditions for about 5 toabout 7 generations; and (c) harvesting resultant seed.
 19. The methodof claim 16, further comprising selecting plants obtained from growingat least one generation of the progeny cotton seed for a desirabletrait.
 20. A method of introducing a desired trait into cotton varietyPHY333WRF, representative seed of the variety having been depositedunder ATCC Accession No. PTA-120742 on Dec. 2, 2013, comprising: (a)crossing PHY333WRF plants with plants of another cotton variety thatcomprise a desired trait to produce F₁ progeny plants; (b) selecting F₁progeny plants that have the desired trait; (c) crossing selectedprogeny plants with PHY333WRF plants to produce backcross progenyplants; (d) selecting for backcross progeny plants that comprise thedesired trait and physiological and morphological characteristics ofcotton variety PHY333WRF; and (e) performing steps (c) and (d) one ormore times in succession to produce selected or higher backcross progenyplants that comprise the desired trait and all of the physiological andmorphological characteristics of cotton variety PHY333WRF listed inTable 1 as determined at the 5% significance level when grown in thesame environmental conditions.